Pesticidal proteins

Details Pesticidal proteins Pesticidal proteins

2000-08-22

2004-01-13

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S419000, C435S418000, C536S023400, C536S023710, C800S302000

Reexamination Certificate

active

06677148

ABSTRACT:

BACKGROUND OF THE INVENTION
Coleopterans are a significant group of agricultural pests which cause extensive damage to crops each year. Examples of coleopteran pests include corn rootworm and alfalfa weevils.
The alfalfa weevil,
Hypera postica
, and the closely related Egyptian alfalfa weevil,
Hypera brunneipennis
, are the most important insect pests of alfalfa grown in the United States, with 2.9 million acres infested in 1984. An annual sum of 20 million dollars is spent to control these pests. The Egyptian alfalfa weevil is the predominant species in the southwestern U.S., where it undergoes aestivation (i.e., hibernation) during the hot summer months. In all other respects, it is identical to the alfalfa weevil, which predominates throughout the rest of the U.S.
The larval stage is the most damaging in the weevil life cycle. By feeding at the alfalfa plant's growing tips, the larvae cause skeletonization of leaves, stunting, reduced plant growth, and, ultimately, reductions in yield. Severe infestations can ruin an entire cutting of hay. The adults, also foliar feeders, cause additional, but less significant, damage.
Approximately 10 million acres of U.S. corn are infested with corn rootworn species complex each year. The corn rootworm species complex includes the northern corn rootworm,
Diabrotica barberi
, the southern corn rootworm,
D. undecimpunctata howardi
, and the western corn rootworm,
D. virgifera virgifera
. The soil-dwelling larvae of these Diabrotica species feed on the root of the corn plant, causing lodging. Lodging eventually reduces corn yield and often results in death of the plant. By feeding on cornsilks, the adult beetles reduce pollination and, therefore, detrimentally affect the yield of corn per plant. In addition, adults and larvae of the genus Diabrotica attack cucurbit crops (cucumbers, melons, squash, etc.) and many vegetable and field crops in commercial production as well as those being grown in home gardens.
Control of corn rootworm has been partially addressed by cultivation methods, such as crop rotation and the application of high nitrogen levels to stimulate the growth of an adventitious root system. However, chemical insecticides are relied upon most heavily to guarantee the desired level of control. Insecticides are either banded onto or incorporated into the soil. Problems associated with the use of some chemical insecticides are environmental contamination and the development of resistance among the treated insect populations.
The soil microbe
Bacillus thuringiensis
(
B.t
.) is a Gram-positive, spore-forming bacterium characterized by parasporal protein inclusions, which can appear microscopically as distinctively shaped crystals. Certain strains of
B.t
. produce proteins that are toxic to specific orders of pests. Certain
B.t
. toxin genes have been isolated and sequenced, and recombinant DNA-based
B.t
. products have been produced and approved for use. In addition, with the use of genetic engineering techniques, new approaches for delivering these
B.t
. endotoxins to agricultural environments are under development, including the use of plants genetically engineered with endotoxin genes for insect resistance and the use of stabilized intact microbial cells as
B.t
. endotoxin delivery vehicles (Gaertner, F. H., L. Kim [1988
] TIBTECH
6:S4-S7). Thus, isolated
B.t
. endotoxin genes are becoming commercially valuable.
Commercial use of
B.t
. pesticides was originally limited to a narrow range of lepidopteran (caterpillar) pests. Preparations of the spores and crystals of
B. thuringiensis
subsp. kurstaki have been used for many years as commercial insecticides for lepidopteran pests. For example,
B. thuringiensis
var. kurstaki HD-1 produces a crystalline &dgr;-endotoxin which is toxic to the larvae of a number of lepidopteran insects.
In recent years, however, investigators have discovered
B.t
. pesticides with specificities for a much broader range of pests. For example, other species of
B.t
., namely israelensis and tenebrionis (a.k.a.
B.t
. M-7, a.k.a.
B.t. san diego
), have been used commercially to control insects of the orders Diptera and Coleoptera, respectively (Gaertner, F. H. [1989] “Cellular Delivery Systems for Insecticidal Proteins: Living and Non-Living Microorganisms,” in
Controlled Delivery of Crop Protection Agents
, R. M. Wilkins, ed., Taylor and Francis, New York and London, 1990, pp. 245-255). See also Couch, T. L. (1980) “Mosquito Pathogenicity of
Bacillus thuringiensis
var. israelensis,”
Developments in Industrial Microbiology
22:61-76; Beegle, C. C., (1978) “Use of Entomogenous Bacteria in Agroecosystems,”
Developments in Industrial Microbiology
20:97-104. Krieg, A., A. M. Huger, G. A. Langenbruch, W. Schnetter (1983)
Z. ang. Ent
. 96:500-508, describe
Bacillus thuringiensis
var. tenebrionis, which is reportedly active against two beetles in the order Coleoptera. These are the Colorado potato beetle,
Leptinotarsa decemlineata
, and
Agelastica alni.
Recently, new subspecies of
B.t
. have been identified, and genes responsible for active &dgr;-endotoxin proteins have been isolated (Höfte, H., H. R. Whiteley [1989
] Microbiological Reviews
52(2):242-255). Höfte and Whiteley classified
B.t
. crystal protein genes into four major classes. The classes were CryI (Lepidoptera-specific), CryII (Lepidoptera- and Diptera-specific), CryIII (Coleoptera-specific), and CryIV (Diptera-specific). The discovery of strains specifically toxic to other pests has been reported. (Feitelson, J. S., J. Payne, L. Kim [1992
] Bio/Technology
10:271-275).
The 1989 nomenclature and classification scheme of Höfte and Whiteley for crystal proteins was based on both the deduced amino acid sequence and the host range of the toxin. That system was adapted to cover fourteen different types of toxin genes which were divided into five major classes. As more toxin-genes were discovered, that system started to become unworkable, as genes with similar sequences were found to have significantly different insecticidal specificities. A revised nomenclature scheme has been proposed which is based solely on amino acid identity (Crickmore et al. [1996] Society for Invertebrate Pathology, 29th Annual Meeting, 3rd International Colloquium on
Bacillus thuringiensis
, University of Cordoba, Cordoba, Spain, September 1-6, abstract). The mnemonic “cry” has been retained for all of the toxin genes except cytA and cytB, which remain a separate class. Roman numerals have been exchanged for Arabic numerals in the primary rank, and the parentheses in the tertiary rank have been removed. Current boundaries represent approximately 95% (tertiary rank), 75% (secondary rank), and 48% (primary rank) sequence identity. Many of the original names have been retained, with the noted exceptions, although a number have been reclassified. See also N. Crickmore, D. R. Zeigler, J. Feitelson, E. Schnepf, J. Van Rie, D. Lereclus, J. Baum, and D. H. Dean (1998), “Revisions of the Nomenclature for the
Bacillus thuringiensis
Pesticidal Crystal Proteins,”
Microbiology and Molecular Biology Reviews
Vol. 62:807-813; and Crickmore, Zeigler, Feitelson, Schnepf, Van Rie, Lereclus, Baum, and Dean, “Bacillus thuringiensis toxin nomenclature” (1999) http://www.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.html. That system uses the freely available software applications CLUSTAL W and PHYLIP. The NEIGHBOR application within the PHYLIP package uses an arithmetic averages (UPGMA) algorithm.
The cloning and expression of a
B.t
. crystal protein gene in
Escherichia coli
has been described in the published literature (Schnepf, H. E., H. R. Whiteley [1981
] Proc. Natl. Acad. Sci. USA
78:2893-2897). U.S. Pat. No. 4,448,885 and U.S. Pat. No. 4,467,036 both disclose the expression of
B.t
. crystal protein in
E. coli.
U.S. Pat. Nos. 4,797,276 and 4,853,331 disclose
B. thuringiensis
strain tenebrionis (a.k.a. M-7, a.k.a.
B.t. san diego
), which can be used to control coleopteran pests in variou

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